The Patch Clamp technique has evolved in recent years from an esoteric method of basic electrophysiological research into properties of cell membranes, into a practical technique for mass screening compounds for potentially therapeutic pharmaceutical properties. The commercial market for such equipment is now in the region of $40M annually. Its role as a tool of basic research has also continued to expand, as a means of discovering types and functions of ion channels through cell membranes, and characterizing their response. Phase I SBIR funding has enabled us to develop a digital clamp amplifier that functions in whole cell clamping and produces waveforms identical to those from comparable analog instruments. We have demonstrated some novel and potentially useful applications, not available with analog devices, such as providing instant "feedback" to a second neuron (dynamic clamp), thus driving one neuron off another's firing rate, and voltage clamping using only a single electrode without an external switch, multiplexing the stimulation and recording stages. This project will lead to a new patch clamp amplifier based on a high-speed digital signal processor to interact with excitable tissues. The instrument integrates the traditional voltage clamp, current clamp, dynamic clamp, and patch clamp into one unit, allowing for transient-free switching among the clamping modes and interactive controls of the neurons. The focus for Phase II includes hardware improvements for patch clamping, software techniques for controls, and experimental validations with mammalian neurons. Patch clamping developed from whole cell voltage clamping, in which measured current is fed back through one of two electrodes in the cell in an amount to keep the voltage across the cell membrane constant, as measured on the second electrode. Ions cross the cell membrane through proteins on the surface which escort them through. These ion channels are frequently voltage gated, open to passing only at certain voltage ranges, or may be chemically gated, thermally gated, or other. Even different voltage gated channels for the same ion may open at different voltages. For patch clamping, the electrode tip is very small, commonly sub-micron, in contact with a part of the cell membrane surface, and a small suction is applied to seal the membrane to the pipette tip. This allows measurement and feedback over only a small bit of membrane, and recording of even single ion channel events. There is a continuing and growing need to catalog all the ion channels across cell membranes, characterize their gate controls, and their function in cell metabolism, and effects on whole animal function and health. A common application is to screen compounds for binding to ion channel proteins to open or close them. Ion channels have become a major target for development of new pharmaceuticals. Variations include voltage clamping, current clamping and conductance clamping. In present application and technology, these 3 techniques each require different analog instruments for the fast responsive feedback needed, and the different parameter measured. Single channel events occur at change rates of up 250K/sec. The high speeds of ion channel events have required analog circuitry. In previous work and publications, it has been shown that a digital throughput of at least 500 KHz would be required to allow a digital instrument to perform as well as the analog instruments in voltage clamping. Very recently, digital signal processing chips (DSP's) operating at as fast as 600 MHz have become commercially available. Digital clamping would save the added cost of A/D and signal conditioning units, and one instrument would be able to perform any of the 4 types of clamping. Other emergent properties allow new avenues of research with digital clamping amplifiers. This proposal is to develop low-noise bi-directional headstages and accessories needed to use this digital clamp amplifier in patch clamping applications, including a chilled headstage to minimize noise, and to develop a library of algorithms for adjustable feedback schemes for different applications, and test the suitability of the instrument for patch clamp experiments. The resulting product is expected to achieve a substantial share of a large market. PUBLIC HEALTH RELEVANCE: Patch clamp technique has emerged as a practical applied technique to test compounds for functional effects on ion channel proteins. Defective ion channels are implicated in several human diseases. The proposed instrument will replace 3 types of analog clamping instruments, and digitizers and signal conditioners with a single, versatile, more powerful digital clamping device capable of serving as a voltage, current or impedance clamp.